Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also require troubleshooting a variety of factors. The tables below highlight common IHC/ICC issues and provide appropriate experimental actions.
Problem: Lack of Staining
||Test or Action|
|Lack of antigen.
||Check protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected).|
|Antibodies do not work due to improper storage.
||Follow storage instructions on the datasheet. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) and avoid repeated freeze-thaw cycles.|
|Inactive primary or secondary antibodies.
||Test reporter system independently to assess reagent viability.|
|Inadequate tissue fixation.
||Try increasing the fixation time or try a different fixative.|
||Reduce the duration of the immersion or post-fixation steps. If immersion fixation cannot be avoided (for example, collection of postmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents.|
|Incompatible secondary and primary antibodies.
||Use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody.|
|Antigen was destroyed before incubation with the primary antibody.
||If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody.|
|Epitope altered during fixation or embedding procedure.
||Try restoring immunoreactivity through various antigen retrieval techniques. |
Embed tissue at 58 °C or below.
|Antigen retrieval was ineffective.
||Increase the time of treatment or change the treatment solution.|
|Reagents omitted or used in wrong order.
||Repeat staining and confirm that correct reagents are used and are added in the correct order.|
Problem: High Background
||Test or Action|
|High concentration of primary and or secondary antibodies.
||Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.|
|Hydrophobic interactions of the antibody and proteins in the tissue.
||Lower the ionic strength of the antibody diluent (particularly monoclonal antibodies respond well to reducing the salt concentration).|
|Non-speci?c binding of primary and/or secondary reagents to tissues.
||Use blocking step just prior to primary antibody incubation (we use 1% bovine serum albumin with 10% normal donkey serum). Non-fat dry milk is another option.|
|Non-specific binding of secondary antibody.
||Use an antibody that has cross-reactive IgG species removed (absorbed against sample species).|
|Tissue dried out.
||Avoid letting the tissue dry during the staining procedure.|
|Reagents sticking to old or poorly prepared slides.
||Start over with freshly prepared or purchased slides.|
|Background due to ionic interactions.
||Increase the ionic strength of the diluent buffer.|
Problem: Cell/Tissue Morphology is Destroyed
||Test or Action|
|Antigen retrieval methods are too harsh.
||Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen.|
|Tissue sections falling off slide. (more common with frozen sections)
||Increase fixation time. Empirically determine an additional or alternative fixative. |
Use freshly prepared, adequately charged slides.
|Tissue section appears torn or folded. Air bubbles under section.
||Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results.|
|Poor resolution of tissue morphology
||Cut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections.|
|Underfixation has physically damaged the tissue or cells during the staining process
||Increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio. |
Cut smaller pieces of tissue for more thorough immersion fixation.
|Autolysis of tissue leading to staining of necrotic debris.
||Increase the fixation time, ratio. Consider using cross-linking fixative.|
Problem: Staining is Inappropriate
||Test or Action |
|Fixation method is inappropriate for the antigen.
||Try a different fixative or increase the fixation time.|
|Antigen retrieval may be inappropriate for this antigen or tissue.
||Try different antigen retrieval conditions.|
|Electrostatic charge of the antigen has been altered.
||Try adjusting the pH or cation concentration of the antibody diluent.|
|Delay in fixation caused diffusion of the antigen.
||Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative.|
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